The neuroprotective effects and possible mechanism of action of a methanol extract from Asparagus cochinchinensis: In vitro and in vivo studies.

Extracts of Asparagus cochinchinensis (AC) have antitumor, anti-inflammatory, and immunostimulant effects. The neurobiological mechanisms underlying the effects of AC have not been sufficiently explored. Thus we performed in vivo and in vitro experiments to further characterize potential therapeutic effects and to clarify the underlying mechanisms. In the tail suspension test immobility time was significantly reduced after administration of AC which suggests antidepressant-like activity without effect on body core temperature. Moreover, in animals pretreated with AC infarct size after occlusion of the middle cerebral artery was reduced. In vitro experiments confirmed neuroprotective effects. Total saponin obtained from AC significantly inhibited H2O2-induced cell death in cultured cortical neurons. The survival-promoting effect by AC saponins was partially blocked by inhibitors for extracellular signal-regulated kinase (ErK) and phosphoinositide 3-kinase Akt (PI3K/Akt) cascades, both of which are known as survival-promoting signaling molecules. Furthermore, phosphorylation of Scr homology-2 (SH2) domain-containing phosphatase 2 (Shp-2) was induced by AC, and the protective effect of AC was abolished by NSC87877, an inhibitor for Shp-2, suggesting an involvement of Shp-2 mediated intracellular signaling in AC saponins. Moreover, AC-induced activation of pShp-2 and ErK1/2 were blocked by NSC87877 indicating that activation of these signaling pathways was mediated by the Shp-2 signaling pathway. These effects appear to be associated with activation of the Shp-2, ErK1/2 and Akt signaling pathways. Our results suggest that AC has antidepressant-like and neuroprotective (reducing infarct size) effects and that activation of pShp-2 and pErK1/2 pathways may be involved in the effects.

Brain-derived neurotrophic factor and glucocorticoids: reciprocal influence on the central nervous system.

Brain-derived neurotrophic factor (BDNF) has multiple roles in the central nervous system (CNS), including maintaining cell survival and regulation of synaptic function. In CNS neurons, BDNF triggers activation of phospholipase Cγ (PLCγ), mitogen-activated protein/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide 3-kinase (PI3K)/Akt pathways, influencing neuronal cells beneficially through these intracellular signaling cascades. There is evidence to suggest that decreased BDNF expression or function is related to the pathophysiology of brain diseases including psychiatric disorders. Additionally, glucocorticoids, which are critical stress hormones, also influence neuronal function in the CNS, and are putatively involved in the onset of depression when levels are abnormally high. In animal models of depression, changes in glucocorticoid levels, expression of glucocorticoid receptor (GR), and alterations in BDNF signaling are observed. Interestingly, several studies using in vivo and in vitro systems suggest that glucocorticoids interact with BDNF to ultimately affect CNS function. In the present review, we provide an overview of recent evidence concerning the interaction between BDNF and glucocorticoids.

Glucocorticoid attenuates brain-derived neurotrophic factor-dependent upregulation of glutamate receptors via the suppression of microRNA-132 expression.

Brain-specific microRNAs (miRs) may be involved in synaptic plasticity through the control of target mRNA translation. Brain-derived neurotrophic factor (BDNF) also contributes to the regulation of synaptic function. However, the possible involvement of miRs in BDNF-regulated synaptic function is poorly understood. Importantly, an increase in glucocorticoid levels and the downregulation of BDNF are supposed to be involved in the pathophysiology of depressive disorders. Previously, we reported that glucocorticoid exposure inhibited BDNF-regulated synaptic function via weakening mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 (MAPK/ERK) and/or phospholipase C-gamma (PLC-gamma) intracellular signaling in cultured neurons [Kumamaru et al (2008) Mol Endocrinol 22:546-558; Numakawa et al (2009) Proc Natl Acad Sci U S A 106:647-652]. Therefore, in this study, we investigate the possible influence of glucocorticoid on BDNF/miRs-stimulated biological responses in cultured cortical neurons. Significant upregulation of miR-132 was caused by BDNF, although miR-9, -124, -128a, -128b, -134, -138, and -16 were intact. Transfection of exogenous ds-miR-132 induced marked upregulation of glutamate receptors (NR2A, NR2B, and GluR1), suggesting that miR-132 has a positive effect on the increase in postsynaptic proteins levels. Consistently, transfection of antisense RNA to inhibit miR-132 function decreased the BDNF-dependent increase in the expression of postsynaptic proteins. U0126, an inhibitor of the MAPK/ERK pathway, suppressed the BDNF-increased miR-132, suggesting that BDNF upregulates miR-132 via the MAPK/ERK1/2 pathway. Interestingly, pretreatment with glucocorticoid (dexamethasone, DEX) reduced BDNF-increased ERK1/2 activation, miR-132 expression, and postsynaptic proteins. We demonstrate that the exposure of neurons to an excess glucocorticoid results in a decrease in the BDNF-dependent neuronal function via suppressing miR-132 expression.

A complex polymorphic region in the brain-derived neurotrophic factor (BDNF) gene confers susceptibility to bipolar disorder and affects transcriptional activity.

Previous studies have suggested that genetic variations in the brain-derived neurotrophic factor (BDNF) gene may be associated with several neuropsychiatric diseases including bipolar disorder. The present study examined a microsatellite polymorphism located approximately 1.0 kb upstream of the translation initiation site of the BDNF gene for novel sequence variations, association with bipolar disorder, and effects on transcriptional activity. Detailed sequencing analysis revealed that this polymorphism is not a simple dinucleotide repeat, but it is highly polymorphic with a complex structure containing three types of dinucleotide repeats, insertion/deletion, and nucleotide substitutions that gives rise to a total of 23 novel allelic variants. We obtained evidence supporting the association between this polymorphic region (designated as BDNF-linked complex polymorphic region (BDNF-LCPR)) and bipolar disorder. One of the major alleles ('A1' allele) was significantly more common in patients than in controls (odds ratio 2.8, 95% confidential interval 1.5-5.3, P=0.001). Furthermore, a luciferase reporter gene assay in rat primary cultured neurons suggests that this risk allele (A1) has a lower-transcription activity, compared to the other alleles. Our results suggest that the BDNF-LCPR is a functional variation that confers susceptibility to bipolar disorder and affects transcriptional activity of the BDNF gene.

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells.

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.

Brain-derived neurotrophic factor enhances depolarization-evoked glutamate release in cultured cortical neurons.

Brain-derived neurotrophic factor (BDNF) has been reported to play an important role in neuronal plasticity. In this study, we examined the effect of BDNF on an activity-dependent synaptic function in an acute phase. First, we found that short-term treatment (10 min) with BDNF enhanced depolarization-evoked glutamate release in cultured cortical neurons. The enhancement diminished gradually according to the length of BDNF treatment. The BDNF-enhanced release did not require the synthesis of protein and mRNA. Both tetanus toxin and bafilomycin abolished the depolarization-evoked glutamate release with or without BDNF, indicating that BDNF acted via an exocytotic pathway. Next, we investigated the effect of BDNF on intracellular Ca(2+). BDNF potentiated the increase in intracellular Ca(2+) induced by depolarization. The Ca(2+) was derived from intracellular stores, because thapsigargin completely inhibited the potentiation. Furthermore, both thapsigargin and xestospongin C inhibited the effect of BDNF. These results suggested that the release of Ca(2+) from intracellular stores mediated by the IP(3) receptor was involved in the BDNF-enhanced glutamate release. Last, it was revealed that the enhancement of glutamate release by BDNF was dependent on the TrkB-PLC-gamma pathway. These results clearly demonstrate that short-term treatment with BDNF enhances an exocytotic pathway by potentiating the accumulation of intracellular Ca(2+) through intracellular stores.

Brain-derived neurotrophic factor triggers a rapid glutamate release through increase of intracellular Ca(2+) and Na(+) in cultured cerebellar neurons.

We reported previously that BDNF induced glutamate release was dependent on intracellular Ca(2+) but not extracellular Ca(2+) in cerebellar neurons (Numakawa et al., 1999). It was revealed that the release was through a non-exocytotic pathway (Takei et al., 1998; Numakawa et al., 1999). In the present study, we monitored the dynamics of intracellular Ca(2+) and Na(+) in cerebellar neurons, and investigated the possibility of reverse transport of glutamate mediated by BDNF. As reported, BDNF increased the intracellular Ca(2+) level. We found that the Ca(2+) increase induced by BDNF was completely blocked by xestospongin C, an IP(3) receptor antagonist, and U-73122, a PLC-gamma inhibitor. Xestospongin C and U-73122 also blocked the BDNF-dependent glutamate release, suggesting that the BDNF-induced transient increase of Ca(2+) through the activation of the PLC-gamma/ IP(3) pathway was essential for the glutamate release. We found that BDNF induced a Na(+) influx. This was blocked by treatment with TTX. U-73122 and xestospongin C blocked the BDNF-induced Na(+) influx, suggesting that the Na(+)influx required the BDNF-induced Ca(2+) increase. Next, we examined the possibility that a co-transporter of Na(+) and glutamate was involved in the BDNF-induced glutamate release. BDNF-induced glutamate release was blocked by L-trans-pyrollidine-2,4-dicalboxylic acid (t-PDC), a glutamate transporter inhibitor, whereas neither the 4-aminopyridine (4AP)- nor high potassium (HK(+))-induced release was blocked by t-PDC. In addition, DL-threo-beta-benzyloxyaspartate (DL-TBOA) also blocked the BDNF-mediated glutamate release, suggesting that reverse transport of glutamate may be involved. All the results therefore suggest that Na(+)-dependent reverse transport contributes to BDNF-mediated transmitter release through the PLC-gamma/IP(3)-mediated Ca(2+) signaling.

Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons.

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.

BDNF rapidly induces aspartate release from cultured CNS neurons.

We investigated the effects of brain-derived neurotrophic factor (BDNF) on aspartate release from cultured cerebellar neurons. This release occurred within 1 min after the addition of 100 ng/ml BDNF. The amount of aspartate released was less than that of glutamate. Aspartate release induced by BDNF was rapid and transient, as in the case of glutamate. Although high potassium evoked the release of both excitatory (glutamate and aspartate) and inhibitory (GABA and glycine) amino acid transmitters, BDNF only induced glutamate and aspartate release. BDNF-induced aspartate release was completely blocked by pretreatment with K252a or TrkB-IgG. The aspartate release induced by BDNF was not dependent on the extracellular Ca(2+), but required intracellular Ca(2+) mobilization. These results showed that BDNF may be involved in excitatory transmission using aspartate as well as glutamate through TrkB-mediated signaling in cerebellum.

Neurotrophin-elicited short-term glutamate release from cultured cerebellar granule neurons.

Brain-derived neurotrophic factor (BDNF) has been suggested to play an important role in neuronal plasticity. In this study, we investigated the effects of BDNF on short-term transmitter release from cultured CNS neurons. Rapid and transient glutamate and aspartate releases induced by BDNF were observed from cultured cortical, hippocampal, striatal and cerebellar neurons. We furthermore investigated the mechanism of release induced by neurotrophins from cerebellar granule cells, since granule cells represent a large homogeneous glutamatergic population. NGF and NT-3 elicited neurotrophin-induced release of glutamate as well as BDNF from the cerebellar granule neurons. The release was dependent on intracellular Ca(2+) mobilization. Pretreatment with K252a and also TrkB-IgG completely blocked the glutamate and aspartate release elicited by BDNF, but not by NGF. The cerebellar granule neurons expressed trkB and p75 mRNAs at high levels, but not trkA mRNA. These results suggested that while BDNF induced release via TrkB, NGF-elicited release was not mediated by Trks. Furthermore, in the experiment using the styryl dye FM1-43, which selectively labels synaptic vesicles, neither BDNF nor NGF evoked dye loss, suggesting that neurotrophin-induced excitatory amino acid release occurs through a non-exocytotic pathway.

Brain-derived neurotrophic factor induces rapid and transient release of glutamate through the non-exocytotic pathway from cortical neurons.

There is increasing interest in the involvement of neurotrophins in neural transmission and plasticity. Thus, we investigated the effects of brain-derived neurotrophic factor (BDNF) on glutamate release from cortical neurons. Treatment of cultured cortical neurons with BDNF induced rapid and transient release of glutamate. This effect was suggested to be mediated by TrkB activation because K252a inhibited the release of glutamate and BDNF phosphorylated TrkB within 30 s. BDNF-induced glutamate release was observed even when using Ca2+-free assay buffer but was inhibited by BAPTA-AM, a cell-permeable Ca2+ chelator. Therefore, BDNF-induced glutamate release was independent of extracelluar Ca2+ but dependent on intracellular Ca2+. Because normal neurotransmitter release is exocytotic, the involvement of the exocytotic pathway in BDNF-induced glutamate release was examined. As botulinum toxin is known to cleave exocytosis-associated proteins, thereby inhibiting exocytosis, it was applied to neurons prior to the release assay. Although botulinum toxin B cleaved VAMP2 and inhibited Ca2+-triggered glutamate release, it did not inhibit the BDNF-induced release of glutamate. These results strongly suggested that BDNF induces rapid and transient release of glutamate from cortical neurons through a non-exocytotic pathway.

Production of reactive oxygen species and release of L-glutamate during superoxide anion-induced cell death of cerebellar granule neurons.

Enhanced production of superoxide anion (O2-) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O2- generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O2- and other ROS and hydroethidine (HEt) specifically for O2- by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O2- and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in L-glutamate release from cerebellar granule neurons. These results indicate that elevation of O2- induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of L-glutamate.

Involvement of phosphatidylinositol-3 kinase in prevention of low K(+)-induced apoptosis of cerebellar granule neurons.

Cerebellar granule neurons obtained from 9-day-old rats die in an apoptotic manner when cultured in serum-free medium containing a low concentration of potassium (5 mM). A high concentration of potassium (26 mM) in the culture medium and BDNF can effectively prevent this apoptosis. The survival effects of high potassium and BDNF were additive, and the effect of high potassium was not blocked by addition of anti-BDNF antibody. These observations indicated that these survival effects were independent. To examine which molecules are involved in the survival pathway induced by BDNF or high K+, we used wortmannin, a specific inhibitor of PI-3 kinase. Wortmannin blocked the survival effects of both BDNF and high K+ on cerebellar granule neurons. Furthermore, in vitro PI-3 kinase assay showed that treatment with BDNF or high K+ induced PI-3 kinase activity, which was diminished by addition of wortmannin. These results indicate that different survival-promoting agents, BDNF and high K+, can prevent apoptosis in cerebellar granule neurons via a common enzyme, PI-3 kinase.

BDNF potentiates spontaneous Ca2+ oscillations in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca2+) levels induced by BDNF using a fluorescence Ca2+ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca2+ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca2+ in only selective proportion of resting neurons without Ca2+ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca2+ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-D-aspartate (NMDA) channel-gated Ca2+ influx via TrkB and regulates the frequency and the amplitude of Ca2+ oscillations. BDNF may have the potential to modulate spontaneous Ca2+ oscillations to regulate neuronal plasticity in developing hippocampal neurons.